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Abstract: RECK (reversion-inducing cysteine-rich protein with Kazal motifs) is a membrane-anchored glycoprotein which regulates MMP2 and MMP9 activity and has been proposed to play a role in embryo implantation while misexpression of RECK has been associated with a variety of carcinomas. Unfortunately, understanding on the steroidal regulation of uterine RECK is lacking. To address this gap in our knowledge, we examined steroidal regulation and cellular expression of Reck mRNA and protein within the mouse uterus in vivo. Uterine Reck mRNA and protein were decreased by estrogen, while progesterone alone had no effect. The estrogen-induced down regulation could be partially blocked by progesterone. RECK was localized primarily to luminal and glandular epithelial cells and the level of expression was regulated in a similar fashion as in whole tissue by the steroids. Knock-down of endogenous RECK in human endometrial epithelial and stromal cells resulted in a significant increase in active MMP9 expression but not that of pro-MMP9 or MMP2. These studies demonstrate that RECK expression in the mouse uterus is steroidally regulated and that within endometrial epithelial and stromal cells, RECK regulates MMP9, but not MMP2 activity.

Abstract: MicroRNAs (miRNAs) are small, non-coding RNA molecules which post-transcriptionally regulate gene expression. We have previously demonstrated that within the uterus, miRNA expression is under steroidal control and that disruption of Dicer1, the enzyme which generates mature miRNAs, leads to abnormalities in the development and function of the female reproductive tract. Despite the apparent importance of miRNAs and the enzymes which lead to their generation, little to no information exists on the mechanisms which regulate the expression of this system in the female reproductive tract. The objective of the current study was to examine steroidal regulation of the miRNAs biogenesis enzymes, Drosha, Dgcr8, Exportin-5 and Dicer1 in the mouse uterus. The results of this study indicate that estrogen and progesterone significantly increased Exportin-5 mRNA expression while only progesterone increased Dicer1 expression. We conclude from these studies that the miRNA biogenesis components Drosha, Dgcr8, Exportin-5 and Dicer1 are expressed in the mouse uterus and that Exportin-5 and Dicer1 appear to be the major steroid regulated components in the miRNA biogenesis pathway. These observations suggest that in addition to steroids modulating miRNA expression at the level of transcription, they may also influence miRNA expression by regulating the expression of the miRNA biogenesis components necessary for their processing to the mature cytoplasmic form.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine which regulates cell proliferation, angiogenesis and immune cell trafficking. Within the uterus, MIF is expressed primarily by uterine epithelial cells in a cycle stage-dependent fashion. Despite characterization of the pattern of uterine MIF expression in a variety of species, virtually no information exists on MIF regulation. As such, the objective of this study was to examine steroidal regulation of Mif in the mouse uterus. Two-month-old, ovariectomized female mice (N=6/treatment group/time point) were treated with estrogen (E2; 1 μg/kg BW) or E2 + progesterone (P4; 2 mg/kg BW) then sacrificed at 0, 4, 8, and 24h post steroid treatment. Uterine Mif was examined by qRT-PCR, Western blot analysis, and immunohistochemical localization. E2 alone, or plus P4, significantly (P<0.05) increased Mif mRNA expression at all time points. Assessment of Mif protein revealed that in contrast to the effect on transcript expression, E2 significantly decreased Mif protein expression by approximately 35% and 65% at 4h and 8h post steroid treatment, respectively, while at 24h, Mif protein levels were approximately 20% below 0h values. Mif localized primarily to luminal and glandular epithelial cells with low stromal expression. The discordant pattern of expression between transcript and protein suggested to us that uterine Mif expression may be regulated by microRNAs (miRs). miR451 is a putative regulator of MIF expression which we have shown to be regulated by E2 within the uterus in a pattern that is inversely correlated to the pattern of Mif uterine protein expression. To test the hypothesis that miR451 regulates Mif protein expression, we transfected human endometrial epithelial cells (HES), with pre-miR-451 precursor, non-targeting precursor (pre-miR-NT; negative control) or transfection buffer alone and assessed miR451 transcript expression by qPCR while MIF protein expression was assessed by Western analysis. Transfection with pre-miR-451 precursor resulted in a significant increase in HES cell expression of mature miR451 which was associated with a significant reduction in MIF protein expression. To further verify that miR451 targets the MIF 3'UTR, HES cells were co-transfected with a Renilla-MIF 3'UTR vector containing the wild-type miR451 seed sequence or a Renilla-MIF 3'UTR mutant vector (mutant miR451 seed sequence), a control vector containing firefly luciferase, and either pre-miR-451 or pre-miR-NT precursors. HES cells co-transfected with constructs which contained the wild-type 3'UTR and pre-miR-451 exhibited significantly less luciferase activity (Renilla normalized to firefly) compared to cells co-transfected with the mutant 3'UTR and pre-miR-451. Cells co-transfected with the mutant 3'UTR and either the pre-miR-451 or pre-miR-NT precursors did not exhibit a significant change in luciferase activity compared to controls. Collectively, these data are interpreted to suggest that miR451 is capable of binding to the 3'UTR of MIF in vitro and suppressing luciferase reporter activity. These observations, coupled with the MIF Western blot data strongly suggest that MIF expression is regulated by miR451. In summary, we demonstrate for the first time that E2 regulates uterine Mif expression and that this regulation appears to be mediated in part by miR451.(platform)

MicroRNAs (miRNAs) are 18 to 25 nucleotide, non-coding RNA molecules which post-transcriptionally regulate gene expression. We have previously demonstrated that estrogen regulates uterine miRNA expression and that disruption of Dicer1, the enzyme which generates mature miRNAs, leads to abnormalities in the development and function of the female reproductive tract. Despite the apparent importance of miRNAs and the enzymes which lead to their generation, little to no information exists on the mechanisms which regulate the expression of this system in the female reproductive tract. The objective of the current study was to examine steroidal regulation of the miRNAs biogenesis enzymes, Drosha, DGCR8, Exportin-5 and Dicer1 in the mouse uterus. To do so, two month-old CD-1 ovariectomized female mice (N=4/treatment group/time point) were treated with estrogen (E2; 1 ug/kg BW), progesterone (P4, 2 mg/kg BW) or E2+P4 (previous doses). Mice were sacrificed at 0, 4, 8 and 24h post steroid treatment. Expression of uterine miRNA biogenesis components was examined by qRT-PCR and localized by immunohistochemistry. Estrogen significantly (P<0.05) increased Exportin-5 mRNA expression at 4h (2.1-fold) and 8h (3-fold) post steroid administration with levels returning to baseline values at 24h. Estrogen did not affect the expression of Drosha, DGCR8 or Dicer1. In a similar manner, P4 increased Exportin-5 expression 1.8- and 2.3-fold at 4h and 8h post treatment, respectively, but had no effect on the mRNA expression of other members of the miRNA biogenesis pathway. In contrast, treatment with both E2 + P4 resulted in a modest (< 1.5-fold increase) but significant (P<0.05) increase in the expression of Drosha, DGCR8 and Dicer1 transcript expression. Exportin-5 was markedly up-regulated by both steroids with a peak 6-fold increase (P<0.01) occurring at 8h post steroid treatment, with expression remaining elevated at 24h post treatment. To verify that these steroid affects were mediated through their respective steroid receptors, a separate group of ovariectomized mice (N=4/treatment group/time point) were challenged with the E2 receptor antagonist ICI 182,780 (ICI; 20 mg/kg BW) and P4 receptor antagonist RU-486 (20 mg/kg BW) and then treated 30 minutes later with the appropriate steroid. ICI blocked the E2-induced increase in Exportin-5 expression while RU-486 blocked the P4-induced increase. These observations confirmed that the stimulatory effects of E2 and P4 on Exportin-5 expression were mediated through their respective receptor pathways. Immunohistochemical localization of Exportin-5 revealed that in 0h controls, Exportin-5 protein expression was low in all uterine cells types. Estrogen administration increased uterine Exportin-5 protein expression with primary localization within the stromal cells and little to no Exportin-5 expression within the epithelium (luminal or glandular). In summary, we demonstrate for the first time that the miRNA biogenesis components Drosha, DGCR8, Dicer1 and Exportin-5 are expressed in the mouse uterus and are regulated by estrogen and progesterone with Exportin-5 and Dicer1 appearing to be the major steroid regulated enzymes in the miRNA biogenesis pathway. These observations suggest that in addition to steroids modulating miRNA expression at the level of transcription, they may also influence miRNA expression by regulating the expression of the miRNA biogenesis enzymes such as Exportin-5 and Dicer1.(poster)